CN108697744B - Phage therapy for pseudomonas infection - Google Patents
Phage therapy for pseudomonas infection Download PDFInfo
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- CN108697744B CN108697744B CN201480058569.1A CN201480058569A CN108697744B CN 108697744 B CN108697744 B CN 108697744B CN 201480058569 A CN201480058569 A CN 201480058569A CN 108697744 B CN108697744 B CN 108697744B
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Abstract
The present invention relates to phage therapy. More specifically, the present invention relates to novel bacteriophages having high specificity against strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa), their manufacture, their components, compositions comprising them and their use in phage therapy.
Description
Technical Field
The present invention relates to novel bacteriophages, compositions comprising said bacteriophages, their manufacture and their use. The invention is particularly useful for treating an infection in a mammal and improving the condition of a subject by modifying the flora in said subject.
Background
Bacteriophages are small viruses that exhibit the ability to infect and kill bacteria while they do not affect cells from other organisms. Phages originally described by William Twort almost a century ago and were discovered independently shortly thereafter by filix d' Herelle, and more than 6000 different phages, including bacteria and archaea (archaeal) viruses, were discovered and described morphologically to date. Most of these viruses are tailed, while a small fraction are polyhedral, filamentous or polymorphic. They can be classified according to their morphology, genetic content (DNA or RNA), specific host, place of life (marine virus or other habitat) and life cycle. As intracellular parasites of bacterial cells, bacteriophages exhibit different life cycles within bacterial hosts: lytic, lysogenic, pseudolysogenic, and chronic infections (Weinbauer, 2004; Drulis-Kawa, 2012). As a normal part of its life cycle, lytic bacteriophages cause lysis of host bacterial cells. Lysogenic phages (also known as temperate phages) can replicate using a lytic life cycle and cause lysis of the host bacteria, or they can incorporate their DNA into the host bacterial DNA and become non-infectious prophages. Regardless of the type of life cycle of the phage, the first step is to attach to receptors on the bacterial cell wall, and then the material of the phage is likely to enter the bacteria. This specific process affects the range of possible phage-bacteria interactions.
Bacteriophages are commonly used as research tools to engineer bacteria in laboratory experiments.
Due to their target host cell specificity, phages have been considered for use as therapies to treat acute and chronic infections, particularly in dermatology, ophthalmology, urology, stomatology, pediatrics, otorhinolaryngology or surgery. However, the concept of the therapeutic use of such bacteriophages to treat bacterial infections has been very controversial from the outset and has not been widely accepted by the public or medical community. Early studies were widely criticized by the lack of suitable controls and inconsistent results. The lack of reproducibility and the many conflicting results obtained in various published studies have led the American Association of medicine and Chemistry (Council on medicine and Chemistry of the American Medical Association) to draw largely contradictory, insufficiently convincing conclusions about the therapeutic value of the lysis filtrate and to recommend further studies to confirm its claimed benefits.
Since the introduction of antibiotics in the 1940 s, little attention has been paid to this field of therapeutics, particularly in the western world. The widespread use of antibiotics has caused the widespread emergence and spread of antibiotic-resistant bacteria worldwide, creating an increasingly serious problem. Therefore, overcoming the limited remaining treatment options to treat the major multidrug resistant microorganisms has become a major therapeutic challenge.
In addition, many pathogenic microorganisms reside within biofilms, which cause additional problems in the design of new antimicrobial agents. In this regard, bacteria that grow as biofilms rather than in single cell ("planktonic") forms tend to be particularly resistant to antimicrobial agents, and the host immune system is particularly difficult to provide a suitable response.
Since the first discovery in the late 19 th century (Fordos 1859), the gram-negative bacterium Pseudomonas aeruginosa (Pseudomonas aeruginosa) has gained a notorious position in the list of notoriously notorious human pathogens (Williams et al, 1894, Freeman et al, 1916). The advent of the antibiotic age has greatly alleviated the previously fatal consequences of acute infections in healthy patients. Only relative progress has been made in the eradication of chronic infections, which occur primarily in individuals with cystic fibrosis or severe burns or immune impairment (Gang et al, 1999, Jones et al, 2010). Two factors that are inherently related in the fatal outcome of infection in these patients are the rapid prescription of inappropriate antibiotic therapy and the occurrence or acquisition of multi-drug resistant strains. Although the use of suitable antibiotics has been reported to be an essential factor in the eradication of p.aeruginosa (Kang et al, 2005, Micek et al, 2005), in contrast, antibiotic abuse significantly contributes to the ability to progressively increase resistance by applying continuous selective pressure to achieve progressively increasing resistance. Antibiotics alone cannot account for the high prevalence of multidrug resistant variants: pseudomonas aeruginosa (p. aeruginosa) has a number of intrinsic resistance mechanisms encoded by chromosomes, including low permeability of the cell envelope and a number of multidrug efflux pumps. Another major factor responsible for the successful invasive behaviour and persistence of this bacterium is its high adaptability, allowing rapid colonization in different environments.
In addition, pathogenic bacteria such as pseudomonas aeruginosa (p. aeruginosa) are capable of forming biofilms, which contribute to their increased resistance to antibiotics. Such biofilms may contain more than one type of bacteria supported and surrounded by secreted extracellular matrix and help the bacteria to colonize various surfaces. Biofilms allow bacteria to attach to surfaces and reach population densities that are otherwise unacceptable, providing increased resistance to not only antibiotics but also to many environmental stresses, including toxins such as heavy metals, bleaches, and other detergents. It is known that bacteria in biofilms can be 100 to 1000 times more resistant to antibiotics than the same bacterial strain grown in planktonic form. This increased resistance means that bacteria apparently susceptible to antibiotics in laboratory tests may be resistant to therapy in a clinical setting. Even if some bacteria are eliminated, the biofilm can provide a resistant reservoir, allowing rapid colonization once the antibiotic is no longer present. Thus, it is clear that biofilms are an important factor in many human diseases. Chemotherapy is not suitable for combating biofilms, as it is precisely where they have evolved to combat. Physical abrasion does provide a means of disrupting biofilm. Unfortunately, many surfaces where biofilms support bacterial pathogenesis, i.e., bones, joints, implanted medical devices, etc., are less suitable for severe abrasion. For example, the surface of a wound or burn is extremely sensitive and delicate. Even where abrasion is both suitable and routinely used, biofilm removal is limited. Oral plaque on the tooth surfaces is a biofilm and is partially removed by daily brushing. However, bacteria remain on the non-brushed surfaces (e.g., in the crevices between teeth) and can quickly and efficiently re-colonize the cleaned surfaces. It is clear from this that the efficacy of the existing methods for removing biofilm is limited.
The rapid adaptation and biofilm-forming ability are the main reasons to identify pseudomonas aeruginosa (p. aeruginosa) as an opportunistic pathogen. They have acquired the status of nosocomial pathogens and can be isolated from clinical samples obtained from wounds, sputum, bladder, urethra, vagina, ear, eye and respiratory tract. The emergence of resistance to the most potent new antibiotics in these clinical pseudomonas aeruginosa (p.aeruginosa) strains, even during treatment, makes combat with the nosocomial pathogens of pseudomonas aeruginosa (p.aeruginosa) a great challenge.
Furthermore, it has been reported that the pathological or physiological condition of a subject is influenced by the microbial balance in the flora of the subject. Therefore, improving the microbial flora or improving the balance or restoring the balance by destroying the pseudomonas aeruginosa (p. aeruginosa) population is also a valuable method of improving the condition of a subject.
There is therefore a great need for new antibacterial agents or compositions that can be used to destroy the strain of pseudomonas aeruginosa (p. aeruginosa), even if the tissue is in a bacterial biofilm, suitable for use in human or animal therapy and suitable for disinfecting materials.
Disclosure of Invention
The present inventors have isolated and characterized novel bacteriophages exhibiting specific lytic activity against Pseudomonas aeruginosa (p. aeruginosa) which are useful as active agents in pharmaceutical or veterinary formulations, in particular for the treatment of Pseudomonas aeruginosa (p. aeruginosa) bacterial infections or for improving microbial balance in a subject. The novel bacteriophages of the invention exhibit strong lytic activity, high selectivity and can be combined to induce controlled destruction of a very large range of pseudomonas aeruginosa (p.
It is an object of the present invention to provide an antibacterial composition comprising at least one, preferably at least two bacteriophages having lytic activity against a strain of Pseudomonas aeruginosa (p.aeruginosa) selected from the group consisting of bacteriophages having a genome comprising SEQ ID NO: 1 to 13 or a sequence having at least 90% identity thereto.
Another object of the invention relates to a bacteriophage which has lytic activity against a strain of Pseudomonas aeruginosa (p. aeruginosa) and the genome of which comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 13 or a sequence having at least 97% identity thereto.
The bacteriophage of the present invention exhibits lytic activity against multidrug resistant strains of pseudomonas aeruginosa (p. aeruginosa), in particular against antibiotic resistant pathogenic strains such as cephalosporinase, carbenicillinase and broad spectrum beta-lactamase (Strateva t. and Yordanov d., 2009).
In another aspect, the invention relates to a bacteriophage having lytic activity against a pathogenic pseudomonas aeruginosa (p.aeruginosa) strain, wherein said bacteriophage is specific for pseudomonas aeruginosa (p.aeruginosa), is active against an antibiotic resistant pseudomonas aeruginosa (p.aeruginosa) strain, and has a productive lytic effect (productive lytic effect) of less than 20.
The invention also relates to an isolated nucleic acid comprised in a bacteriophage of the invention, preferably comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 13 or a sequence having at least 97% identity thereto; and an isolated polypeptide encoded by the nucleic acid.
Another object of the invention is a composition comprising a nucleic acid or polypeptide as defined above.
The compositions of the present invention will also typically comprise a pharmaceutically or veterinarily acceptable excipient or carrier. They may be liquid, semi-liquid, solid or freeze-dried.
Another object of the invention relates to a bacteriophage, nucleic acid, polypeptide or composition as defined above for use in treating an infection in a mammal, modifying the microbial flora in a mammal, disinfecting a material, and/or killing pseudomonas aeruginosa (p.
The invention also relates to the use of one or several lytic bacteriophages for improving the condition of a subject by improving the microbial flora in said subject. The microbial flora may be improved by correcting, altering or restoring the proper balance of microorganisms in the flora.
The present invention also relates to a method for treating an infection in a mammal, said method comprising administering to said mammal at least one bacteriophage, nucleic acid, polypeptide or composition as defined above.
The invention also relates to a method for treating a surface or material suspected of being contaminated with pseudomonas aeruginosa (p. aeruginosa) bacteria, said method comprising applying to said surface or material at least one bacteriophage, nucleic acid, polypeptide or composition as defined above. The surface or material may be any device, surface of a container or laboratory material, clothing, etc.
Another object of the invention relates to a kit comprising a composition as defined above and a mechanism for applying said composition to an object or surface.
Another object of the invention relates to a method for predicting or determining the efficacy of phage therapy in a subject, wherein said method comprises determining in vitro the lytic activity of one or more phages of the invention against a strain of pseudomonas aeruginosa (p.aeruginosa) from a sample of said subject, the lytic activity of one or more phages of the invention against at least one strain of pseudomonas aeruginosa (p.aeruginosa) from said sample being indicative of an effective treatment. The method also optionally includes the step of treating the subject with at least one bacteriophage having lytic activity to a pseudomonas aeruginosa (p.
In another aspect, the invention provides a method of selecting a subject or determining whether a subject is susceptible to benefit from phage therapy, wherein said method comprises the step of determining in vitro the lytic activity of one or more bacteriophage of the invention against a pseudomonas aeruginosa (p.aeruginosa) strain from a sample of said subject, the lytic activity of one or more of said bacteriophage against at least one pseudomonas aeruginosa (p.aeruginosa) strain being indicative of a responder subject.
The invention can be used in any mammal, preferably in humans, or for the treatment of any material, including laboratory materials or medical devices.
Drawings
FIG. 1: in vitro efficacy of the bacteriophage of the present invention on various combinations of pseudomonas aeruginosa (p.
FIG. 2: the in vivo efficacy of the bacteriophage of the present invention on various combinations of pseudomonas aeruginosa (p.
FIG. 3: in vivo efficacy of the bacteriophage of the present invention against Is580 pseudomonas aeruginosa (p. aeruginosa) strain mediated infection.
Detailed Description
The present invention relates to novel bacteriophages, their components, compositions comprising said bacteriophages, their manufacture and their use as antibacterial agents, in particular for treating infections in mammals and for improving the condition of a subject by modifying the microbial flora in said subject.
Definition of
To facilitate an understanding of the present invention, a number of terms are defined below.
As used herein, the term "bacteriophage" refers to a functional bacteriophage particle comprising a nucleic acid genome packaged in a protein envelope or capsid. The term also refers to portions of the phage, including, for example, the head; or an assembly of phage components providing substantially the same functional activity.
The term "phenotypic characteristic" more preferably refers to the morphology and/or host range of the bacteriophage. Methods for determining a bacteriophage phenotype are known per se in the art and include, for example, determining bacterial host range and/or activity against a biofilm produced by certain bacterial strains.
The term "lytic activity" as used in the present invention refers to the property of a bacteriophage to cause lysis of bacterial cells. The lytic activity of the phages can be tested on a strain of pseudomonas aeruginosa (p. aeruginosa) according to techniques known per se in the art (see also the experimental part).
The term "variant" of a reference bacteriophage refers to a bacteriophage that has a variation in the genomic sequence and/or polypeptide encoded thereby as compared to the reference bacteriophage, while retaining the same phenotypic characteristics as the reference bacteriophage. The variants typically comprise, for example, silent mutations, conservative mutations, minor deletions, and/or minor duplications of the genetic material, and retain the phenotypic characteristics of the reference phage. In a preferred embodiment, the variant of the invention retains any observable feature or characteristic depending on the genome of the bacteriophage of the invention, i.e. the phenotypic characteristics of said bacteriophage, and/or the lytic activity against a strain of pseudomonas aeruginosa (p. Preferred variants have less than 5% nucleic acid variation, even more preferably less than 4%, more preferably less than 2% compared to the genome of the reference phage. Alternatively or in combination, the variant preferably has less than 5% amino acid variation in the encoded polypeptide sequence compared to the polypeptide of a reference phage.
The term "% identity" in the context of nucleic acid or amino acid sequences refers to the level of identity or homology between the sequences and can be determined by techniques known per se in the art. Typically, the% identity between two nucleic acid or amino acid sequences is determined using Computer programs, such as GAP provided in the GCG Package (Program Manual for the Wisconsin Package), version 8,1996, 8 months, the Genetics Computer Group (Genetics Computer Group),575Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D. (1970), Journal of Molecular Biology,48, 443-. Using settings adjusted to, for example, DNA sequences (specifically: a GAP creation penalty of 5.0 and a GAP extension penalty of 0.3), nucleic acid molecules can be aligned to each other using Pileup alignment software available as part of the GCG package. The% identity between two sequences refers to the identity over the entire length of the sequences.
The term "fragment" of a nucleic acid generally refers to a fragment having at least 10 contiguous nucleotides of the nucleic acid, more preferably at least 15, 20, 25, 30, 35, 40, 50 or more contiguous nucleotides of the nucleic acid.
The term "fragment" of a polypeptide generally refers to a fragment having at least 5 contiguous amino acids of the polypeptide, more preferably at least 10, 15, 20, 30, 40, 50 or more contiguous amino acids of the polypeptide.
The term "ESBL pseudomonas aeruginosa (p. aeruginosa) strain" refers to a strain of pseudomonas aeruginosa (p. aeruginosa) that produces a cephalosporin enzyme and/or a broad spectrum of beta-lactamase enzymes, including various forms of antibiotic resistance such as AmpC beta-lactamase or class a carbenicillin-hydrolyzing beta-lactamase enzymes and the like.
The term "specific" or "specificity" with respect to a bacteriophage refers to the type of host that the bacteriophage is capable of infecting. Specificity is usually mediated by the tail fibers of the phage, which are involved in the interaction with receptors expressed on the cells. A bacteriophage that is "specific" for pseudomonas aeruginosa (p.aeruginosa) more preferably refers to a bacteriophage that can infect one or several strains of pseudomonas aeruginosa (p.aeruginosa) and that is incapable of infecting a non-pseudomonas aeruginosa bacterium under physiological conditions.
As used herein, the term "polypeptide" refers to a polypeptide of any size, including, for example, small peptides of 5 to 20 amino acids, longer polypeptides, proteins, or fragments thereof.
The term "PLE" or "productive lysis effect" refers to the ratio between the amount released (burst) of a given bacteriophage and the productive lysis time. The release amount and productive lysis time are parameters defining the phage-host interaction and correspond to the average yield of phage particles produced by infection of a bacterium with a phage and the time taken for free phage to lyse a bacterial cell, respectively.
In the context of the present specification, the term "isolated phage" should be taken to mean material removed from its naturally occurring original environment. In the case of bacteriophages, the term refers specifically to bacteriophages that have been cultured, purified, and/or cultured separately from the environment in which they are naturally found, for example. The term "isolated" with respect to a nucleic acid or polypeptide refers to a nucleic acid molecule or polypeptide that is separated from at least some components of its natural environment, such as proteins, lipids, and/or nucleic acids, for example.
As used herein, the term "pharmaceutically or veterinarily acceptable" refers to any material (e.g., carrier, excipient, or vehicle) that is compatible for use in a mammalian subject. These materials include physiologically acceptable solutions or media that are not harmful to the organism or cause any significant specific or non-specific immune response or abrogate the biological activity of the active compound. For formulation of the compositions into liquid preparations, saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof may be used as pharmaceutically or veterinarily acceptable excipients or carriers. Other conventional additives such as thickeners, diluents, buffers, preservatives, surfactants, antioxidants and bacteriostats may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to the composition to prepare injectable preparations such as aqueous solutions, suspensions and emulsions, oral preparations such as pills, capsules, granules or tablets, or powder preparations.
As used herein, "PFU" refers to a plaque forming unit, as is well defined in the art. Lytic phages lyse the host cells, producing a clear zone (or plaque) on the culture plate. Theoretically, each plaque is formed by one phage, and the number of plaques multiplied by the dilution factor equals the total number of phage in the test preparation.
The term "treatment" or "therapy" refers to the curative or prophylactic treatment of a disease. Curative treatment is defined as treatment that causes the disease to heal, or treatment that directly or indirectly alleviates, stabilizes or eliminates the symptoms of the disease or the pain it causes, or treatment that improves the condition of the subject or slows the progression of the disease. Prophylactic treatment encompasses both treatment that results in the prevention of disease and treatment that reduces and/or delays the incidence of disease or the risk of its occurrence.
The term "mammal" includes human subjects as well as non-human mammals such as pets (e.g., dogs, cats), horses, ruminants, sheep, goats, pigs, and the like.
As used herein, the term "biofilm" refers to a heterogeneous formation of bacteria growing on a variety of different surfaces; preferably a bacterial community grown embedded in a exopolysaccharide matrix attached to a solid biological or non-biological surface.
As used herein, the term "compromise" refers to any change in integrity. Damaging a bacterial biofilm is understood to be the invasion of the biofilm by phages, infection of the biofilm-associated bacteria or lysis thereof, and/or partial or total removal of the biofilm (i.e. by stopping colonization and/or disrupting the biofilm).
As used herein, the term "sample" refers to any sample containing cells. Examples of such samples include fluids such as blood, plasma, saliva or urine, as well as biopsy, organ, tissue or cell samples. The sample may be treated prior to use.
As used herein, the term "subject" or "patient" refers to an animal, preferably a mammal, even more preferably a human, including adults and children. However, the term "subject" also encompasses non-human animals, especially mammals such as dogs, cats, horses, cows, pigs, sheep, and non-human primates, and the like.
As used herein, the term "efficacy" of a treatment or "response" to phage therapy refers to a treatment that results in a reduction in the number of pseudomonas aeruginosa (p.aeruginosa) strains in a subject following phage treatment compared to the number of p.aeruginosa strains prior to treatment. A "good responder" subject is a subject that shows or will show clinically significant recovery when treated with phage therapy.
The term "mixture" or composition of bacteriophages refers to a combination of different types of bacteriophages. The bacteriophages in the mixture/composition are preferably formulated together, i.e., in the same container or package, although they may also be used as a kit of parts, where the bacteriophages (or some bacteriophages) are formulated or packaged separately and combined at the time of use or administration.
Description of the embodiments
The present invention relates to novel phage therapies. More particularly, the present invention relates to novel bacteriophages having high specificity for strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa), their manufacture, their components, compositions comprising them and their use in phage therapy.
Phage display:
in a first aspect, the present invention discloses the isolation and characterization of novel phage specific for the pseudomonas aeruginosa (p. aeruginosa) strain and displaying, alone or in combination, an attractive host range of lytic activity. These phages were selected from environmental samples, isolated, sequenced and characterized. As indicated, the phages individually and in combination have activity against a strain of pseudomonas aeruginosa (p. They are significantly effective against pathogenic pseudomonas aeruginosa (p. aeruginosa) strains such as antibiotic resistant pseudomonas aeruginosa (p. aeruginosa) strains, for example ESBL pseudomonas aeruginosa (p. aeruginosa) strains. Furthermore, the bacteriophage of the present invention has a significant productive lytic effect ("PLE") below 20, more preferably below 15, still more preferably between 0.3 and 15. Furthermore, the bacteriophages of the invention are specific for strains of pseudomonas aeruginosa (p. aeruginosa), i.e. they do not cause lysis of non-pseudomonas aeruginosa bacteria. As will be further explained, the present invention shows that these bacteriophages may be combined and formulated in a state suitable for use as pharmaceutical or veterinary agents to exhibit a targeted and very strong antibacterial effect against a controlled range of strains of pseudomonas aeruginosa (p.
More specifically, the following phages have been selected and characterized. Their corresponding nucleic acid sequences are also noted.
TABLE 1
| SEQ ID No. | Bacteriophage |
| SEQ ID NO:1 | BP1384 |
| SEQ ID NO:2 | BP1429 |
| SEQ ID NO:3 | BP1430 |
| SEQ ID NO:4 | BP1433 |
| SEQ ID NO:5 | BP1450 |
| SEQ ID NO:6 | BP1644 |
| SEQ ID NO:7 | BP1647 |
| SEQ ID NO:8 | BP1648 |
| SEQ ID NO:9 | BP1649 |
| SEQ ID NO:10 | BP1650 |
| SEQ ID NO:11 | BP1658 |
| SEQ ID NO:12 | BP1661 |
| SEQ ID NO:13 | BP1662 |
Lysis of these phages has been determined on a wide variety of pseudomonas aeruginosa (p. aeruginosa) strains. As disclosed in the table below, these phages were selected for their potency and combinatorial potential. In this table, the lytic effect of the phage on the reference and pathogen resistant strains is demonstrated to demonstrate high lytic potential.
TABLE 2
| Bacteria/bacteriophages | 1384 | 1429 | 1430 | 1433 | 1450 | 1644 | 1647 | 1648 | 1649 | 1650 | 1658 | 1661 | 1662 |
| LMG 24882 | + | + | + | + | + | + | + | + | + | + | + | + | |
| LMG 24883 | + | +/- | + | + | + | + | + | + | + | + | + | ||
| LMG 24886 | + | + | + | + | +/- | + | +/- | + | |||||
| LMG 24887 | +/- | +/- | +/- | + | + | +/- | + | + | +/- | +/- | |||
| LMG 24891 | + | + | + | + | + | + | + | + | + | + | + | + | |
| LMG 24892 | + | + | + | + | + | + | + | + | +/- | + | + | ||
| LMG 24893 | + | + | + | + | + | + | + | + | + | + | + | + | + |
| LMG 24896 | + | + | + | + | + | + | + | + | + | + | + | + | + |
| LMG 24898 | + | + | + | + | + | + | + | + | + | + | + | + | + |
| LMG 24901 | +/- | + | +/- | ||||||||||
| LMG 24903 | +/- | +/- | + | + | +/- | ||||||||
| LMG 24904 | +/- | +/- | +/- | + | +/- | ||||||||
| LMG 24905 | +/- | + | + | + | + | + | |||||||
| LMG 24909 | + | +/- | + | + | + | + | + | + | +/- | + | |||
| LMG 24913 | +/- | + | + | +/- | +/- | ||||||||
| LMG 24916 | + | + | + | + | + |
Further results on highly resistant strains from wounds or burns are presented below, further confirming the dramatic activity profile of the phages of the invention and their complementarity.
TABLE 3
| 1384 | 1429 | 1430 | 1433 | 1450 | 1644 | 1647 | 1648 | 1649 | 1650 | 1658 | 1661 | 1662 | CAR* | |
| LMG 25000 | + | + | + | - | + | + | + | - | + | - | + | + | - | 1 |
| LMG 25122 | - | - | - | - | + | + | - | - | + | - | + | + | - | 5 |
| LMG 25140 | - | + | - | - | + | + | - | - | + | - | + | + | + | 5 |
| LMG 25133 | - | - | - | + | + | - | + | - | - | - | + | + | - | 2 |
| LMG 25165 | - | - | + | - | - | - | + | - | - | - | - | + | - | 5 |
| LMG 25146 | - | - | - | - | + | + | - | - | + | - | + | + | - | 4 |
CAR: ATB resistance type
As can be seen from tables 2 and 3, the phages individually have a very strong lytic capacity and can produce a combination (or mixture) of these phages capable of killing all the pseudomonas aeruginosa (p. aeruginosa) strains tested, thus producing a broad spectrum antibacterial composition.
As an example, a mixture of all 13 bacteriophages according to the invention was able to effectively kill all bacteria listed in table 2 and table 3.
In addition, the specificity of the phage has been tested on a number of non-P.aeruginosa strains. More specifically, the experimental part demonstrates that the bacteriophage of the present invention has no lytic effect on any bacterium selected from the group consisting of Escherichia coli (Escherichia coli), Acinetobacter baumannii (Acinebacter baumannii), Enterobacter aerogenes (Enterobacter aeogens), Enterobacter albugineae (Enterobacter albureae), Enterobacter cloacae (Enterobacter cloacae), Klebsiella pneumoniae (Klebsiella pneumoniae), Proteus mirabilis (Porteus), Staphylococcus aureus (Staphylococcus aureus), Stenotrophomonas maltophilia (Stenotrophia maltophilia) and/or Serratia marcescens (Serratia marcocens).
A particular object of the present invention is therefore a bacteriophage with lytic activity against a strain of pseudomonas aeruginosa (p. aeruginosa), the genome of said bacteriophage comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 13 or a sequence having at least 97% identity thereto, preferably at least 98% or 99% identity thereto.
As will be disclosed in more detail below, the bacteriophage of the present invention may be cultured, amplified, isolated, purified and used in phage therapy for, for example, Pseudomonas aeruginosa-mediated disorders. Furthermore, variants of these phages that retain the phenotype (e.g. specificity and lytic activity) of the phage can be produced and/or isolated by techniques known per se in the art.
The phage of the present invention can be prepared by standard culturing, isolation and purification methods. For example, Pseudomonas aeruginosa producing bacteria are cultured, infected with a bacteriophage sample, and then treated to remove bacterial cells and debris. The enriched phage solution can be plated in a medium, such as agar medium, with the embedded pseudomonas aeruginosa susceptible host strain to obtain plaques. Individual plaques can then be picked for subsequent phage purification and amplification. One or more selective amplification cycles of the bacteriophage of the present invention may be performed, for example, as follows: the phage was mixed with competent pseudomonas aeruginosa, then growth medium was added and incubated under the selected experimental growth conditions. After centrifugation, the clarified amplification supernatant is filtered through a filter and subjected to another selective amplification cycle or tested for the presence of lytic activity.
The titer of the phage in suspension and visualization of the plaque morphology of the phage of the invention can then be assessed by known methods, for example by plaque counting. Furthermore, as is well known in the art, the bacteriophages of the present invention can be processed by any suitable method into various forms (liquid, freeze-dried, etc.) for short-term, long-term, frozen or any other type of storage (see, e.g., Clark, 1962).
The activity of the bacteriophage of the present invention can be evaluated by a method known in the art, such as a plaque assay (also called double agar method), on the basis of: the phages were grown using potential host cells and then evaluated for their ability to kill the host bacterial cells. In the plaque assay, after incubation in soft agar medium for a period of time, the phage causes lysis of the target pseudomonas aeruginosa strain, producing a clearing zone on the plate called a plaque.
In a particular embodiment, the invention relates to a BP1384 bacteriophage or any variant thereof. BP1384 phage, or any variant thereof, can be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1384 or any variant thereof is specific for and has lytic activity against strains LMG24882, LMG24883, LMG24886, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898 and/or LMG 24909. The genome of BP1384 comprises the nucleotide sequence set forth in SEQ ID NO: 1 or a sequence identical to SEQ id no: 1, more preferably at least 85% identity, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from a BP1384 bacteriophage or variant thereof. The invention also encompasses an isolated polypeptide encoded by a BP1384 bacteriophage or variant thereof or encoded by an isolated nucleic acid sequence derived from a BP1384 bacteriophage of the invention. Another feature of the BP1384 bacteriophage of the present invention is that the PLE is less than 20, more preferably less than 15, and still more preferably around 6.2.
In another particular embodiment, the invention relates to a BP1429 bacteriophage or any variant thereof. The BP1429 phage, or any variant thereof, may be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1429 or any variant thereof is specific for and has lytic activity against the strains LMG24882, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898 and/or LMG 24916. The genome of BP1429 comprises the nucleotide sequence set forth in SEQ ID NO: 2 or a sequence identical to SEQ ID NO: 2, more preferably at least 85%, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1429 phage or variant thereof. The invention also encompasses isolated polypeptides encoded by a BP1429 bacteriophage or variant thereof or encoded by an isolated nucleic acid sequence from a BP1429 bacteriophage of the invention. Another feature of the BP1429 bacteriophage of the present invention is that PLE is less than 20, more preferably less than 15, still more preferably around 0.70.
In another aspect, the invention relates to a BP1430 bacteriophage or any variant thereof. BP1430 phage or any variant thereof can be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1430 or any variant thereof is specific for and has lytic activity against strains LMG24882, LMG24883, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898, LMG24901 and/or LMG 24918. The genome of BP1430 comprises the nucleotide sequence set forth as SEQ ID NO: 3 or a sequence identical to SEQ ID NO: 3, more preferably at least 85%, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Isolated nucleic acid sequences from the BP1430 phage or variants thereof are also provided. The invention also encompasses isolated polypeptides encoded by a BP1430 bacteriophage or variant thereof or encoded by an isolated nucleic acid sequence from a BP1430 bacteriophage of the invention. Another feature of the BP1430 bacteriophage of the invention is that the PLE is less than 20, more preferably less than 15, and still more preferably around 3.
In another aspect, the invention relates to a BP1433 phage or any variant thereof. BP1433 phage, or any variant thereof, can be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1433 or any variant thereof is specific for and has lytic activity against strain LMG24882, LMG24883, LMG24886, LMG24887, LMG24891, LMG24892, LMG24893, LMG24896, LMG24905, LMG24909 and/or LMG 24916. The genome of BP1433 comprises the nucleotide sequence as set forth in SEQ ID NO: 4 or a sequence identical to SEQ ID NO: 4, more preferably at least 85%, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1433 phage or variant thereof. The invention also encompasses an isolated polypeptide encoded by a BP1433 phage or variant thereof or by an isolated nucleic acid sequence from a BP1433 phage of the invention. Another feature of the BP1433 phage of the present invention is that the PLE is below 20, more preferably below 15, still more preferably around 4.
In another particular embodiment, the invention relates to a BP1450 bacteriophage or any variant thereof. The BP1450 phage, or any variant thereof, may be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1450 or any variant thereof is specific for and has lytic activity against strains LMG24882, LMG24883, LMG24886, LMG24887, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898, LMG24903, LMG24904, LMG24905, LMG24909 and/or LMG 24913. The genome of BP1450 comprises the nucleotide sequence set forth in SEQ ID NO: 5 or a sequence identical to SEQ id no: 5, more preferably at least 85% identity, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1450 phage or variant thereof. The present invention also encompasses isolated polypeptides encoded by the BP1450 phage or variants thereof or encoded by isolated nucleic acid sequences from the BP1450 phage of the present invention. Another feature of the BP1450 bacteriophage of the present invention is that the PLE is below 20, more preferably below 15, still more preferably around 2.
In another aspect, the invention relates to a BP1644 bacteriophage or any variant thereof. The BP1644 bacteriophage, or any variant thereof, may be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1644 or any variant thereof is specific for and has lytic activity against strain LMG24882, LMG24883, LMG24886, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898, LMG24905 and/or LMG 24909. The genome of BP1644 comprises the nucleotide sequence set forth in SEQ ID NO: 6 or a sequence identical to SEQ id no: 6, more preferably at least 85% identity, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1644 bacteriophage or a variant thereof. The invention also encompasses isolated polypeptides encoded by a BP1644 bacteriophage or a variant thereof or encoded by an isolated nucleic acid sequence from a BP1644 bacteriophage of the invention. The BP1644 bacteriophage of the invention is further characterized by a PLE below 20, more preferably below 15, still more preferably around 1.5.
In another particular embodiment, the invention relates to a BP1647 bacteriophage or any variant thereof. The BP1647 bacteriophage, or any variant thereof, may be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1647 or any variant thereof is specific for and has lytic activity against strains LMG24882, LMG24883, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898, LMG24903 and/or LMG 24916. The genome of BP1647 comprises the nucleotide sequence set forth in SEQ ID NO: 7 or a sequence identical to SEQ id no: 7, more preferably at least 85% identity, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1647 phage or variants thereof. The invention also encompasses isolated polypeptides encoded by a BP1647 bacteriophage or a variant thereof or encoded by an isolated nucleic acid sequence from a BP1647 bacteriophage of the invention. Another feature of the BP1647 bacteriophage of the invention is that the PLE is lower than 20, more preferably lower than 15, still more preferably around 0.4.
In another particular embodiment, the invention relates to a BP1648 bacteriophage or any variant thereof. The BP1648 phage or any variant thereof may be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1648 or any variant thereof is specific for and has lytic activity against strain LMG24882, LMG24883, LMG24891, LMG24893, LMG24896, LMG24898 and/or LMG 24909. The genome of BP1648 comprises the nucleotide sequence set forth in SEQ ID NO: 8 or a sequence identical to SEQ ID NO: 8, more preferably at least 85%, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1648 phage or variants thereof. The invention also encompasses isolated polypeptides encoded by a BP1648 bacteriophage or a variant thereof or encoded by an isolated nucleic acid sequence from a BP1648 bacteriophage of the invention. Another feature of the BP1648 bacteriophage of the invention is that the PLE is lower than 20, more preferably lower than 15, still more preferably around 2.
In another aspect, the invention relates to a BP1649 bacteriophage or any variant thereof. The BP1649 phage or any variant thereof may be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1649 or any variant thereof is specific for and has lytic activity against strains LMG24882, LMG24883, LMG24886, LMG24887, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898, LMG24905, LMG24909 and/or LMG 24913. The genome of BP1649 comprises the nucleotide sequence set forth in SEQ ID NO: 9 or a sequence identical to SEQ ID NO: 9, more preferably at least 85% identity, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1649 phage or variants thereof. The invention also encompasses isolated polypeptides encoded by a BP1649 bacteriophage or a variant thereof or encoded by an isolated nucleic acid sequence from a BP1649 bacteriophage of the invention. Another feature of the BP1155 bacteriophage of the present invention is that the PLE is less than 20, more preferably less than 15, still more preferably around 3.5.
In another particular embodiment, the invention relates to a BP1650 bacteriophage or any variant thereof. The BP1650 bacteriophage or any variant thereof may be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1650 or any variant thereof is specific for and has lytic activity against the strains LMG24882, LMG24893, LMG24896, LMG24898, LMG24905 and/or LMG 24909. The genome of BP1650 comprises the nucleotide sequence set forth as SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10, more preferably at least 85%, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from a BP1650 bacteriophage or variant thereof. The invention also encompasses isolated polypeptides encoded by a BP1650 bacteriophage or variant thereof or encoded by an isolated nucleic acid sequence from a BP1650 bacteriophage of the invention. Another feature of the BP1650 bacteriophage of the invention is that the PLE is below 20, more preferably below 15, still more preferably around 14.
In another aspect, the invention relates to the BP1658 bacteriophage or any variant thereof. The BP1658 phage, or any variant thereof, can be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1658 or any variant thereof is specific for and has lytic activity against strains LMG24882, LMG24887, LMG24891, LMG24893, LMG24896 and/or LMG 24898. The genome of BP1658 comprises the nucleotide sequence set forth as SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11, more preferably at least 85%, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Also provided is an isolated nucleic acid sequence from the BP1658 bacteriophage or a variant thereof. The invention also encompasses isolated polypeptides encoded by the BP1658 bacteriophage or a variant thereof, or encoded by an isolated nucleic acid sequence from the BP1658 bacteriophage of the invention. Another feature of the BP1658 bacteriophage of the present invention is that the PLE is less than 20, more preferably less than 15, still more preferably around 3.
In another aspect, the invention relates to a BP1661 bacteriophage or any variant thereof. The BP1661 phage, or any variant thereof, can be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1661 or any variant thereof is specific for and has lytic activity against strains LMG24882, LMG24883, LMG24886, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898 and/or LMG 24909. The genome of BP1661 comprises the nucleotide sequence set forth in SEQ ID NO: 12 or a sequence identical to SEQ ID NO: 12, more preferably at least 85% identity, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Isolated nucleic acid sequences from the BP1661 phage or variants thereof are also provided. The invention also encompasses isolated polypeptides encoded by a BP1661 bacteriophage or variant thereof, or by an isolated nucleic acid sequence from a BP1661 bacteriophage of the invention. Another feature of the BP1661 phage of the invention is that the PLE is below 20, more preferably below 15, still more preferably around 4.
In another aspect, the invention relates to a BP1662 bacteriophage or any variant thereof. The BP1662 phage, or any variant thereof, can be produced or amplified, for example, in pseudomonas aeruginosa strain PAO 1. BP1662 or any variant thereof is specific for and has lytic activity against the strains LMG24883, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898 and/or LMG 24916. The genome of BP1662 comprises the nucleotide sequence set forth in SEQ ID NO: 13 or a sequence identical to SEQ ID NO: 13, more preferably at least 85%, still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity. Isolated nucleic acid sequences from the BP1662 phage or variants thereof are also provided. The invention also encompasses isolated polypeptides encoded by a BP1662 bacteriophage or variant thereof, or encoded by an isolated nucleic acid sequence from a BP1662 bacteriophage of the invention. Another feature of the BP1662 phage of the invention is that the PLE is below 20, more preferably below 15, still more preferably around 1.
Nucleic acids and polypeptides
The present invention relates to a nucleic acid comprised in a bacteriophage of the present invention or any fragment of such a nucleic acid. The term fragment more preferably refers to a fragment comprising (or consisting of) an open reading frame. The nucleic acid may be DNA or RNA, single-stranded or double-stranded.
The nucleic acid may be isolated from the deposited phage or produced using recombinant DNA techniques (e.g., Polymerase Chain Reaction (PCR) amplification, cloning), enzymatic synthesis, or chemical synthesis, or a combination thereof, according to common techniques known per se in the art. Also included are homologous sequences and fragments thereof, including but not limited to natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted and/or inverted.
In a particular embodiment, the present invention relates to a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-13 or a sequence identical to SEQ ID NO: 1-13, a sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity.
In another particular embodiment, the invention relates to a nucleic acid comprising the sequence of the fragment: selected from the group consisting of SEQ ID NO: 1-13 or a fragment of a sequence identical to any one of SEQ ID NOs: 1-13, comprising an open reading frame or a regulatory element such as a promoter.
The nucleic acids of the invention may be in free form or cloned in vectors.
In another aspect, the invention also relates to an isolated polypeptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: 13 is encoded by the nucleic acid sequence of seq id no. The polypeptides may be produced by techniques known per se in the art, such as synthesis, recombinant techniques, or combinations thereof. The polypeptides may be isolated or purified, used as antibacterial agents or as reagents for in vitro assays.
Compositions of the invention
One aspect of the present invention relates to a composition comprising at least one, more preferably at least two or more bacteriophages as described above, and optionally pharmaceutically or veterinarily acceptable excipients. As described, the bacteriophage of the present invention has very strong lytic activity against pseudomonas aeruginosa (p. Combinations of these phages can be generated to extend the host range and produce highly effective antibacterial compositions.
More specifically, the present invention relates to an antibacterial composition comprising at least two bacteriophages having lytic activity against a strain of Pseudomonas aeruginosa (p.aeruginosa) selected from the group consisting of bacteriophages having genomes comprising SEQ ID NO: 1 to 13 or a sequence which is at least 90% identical thereto, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
In a preferred embodiment, the composition of the invention comprises at least three, even more preferably at least four, different bacteriophages selected from the group consisting of bacteriophages whose genomes comprise SEQ ID NO: 1 to 13 or a sequence which is at least 90% identical thereto, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto. The composition of the invention may comprise at least 5, 6, 7, 8, 9, 10, 11, 12 or all 13 different types of bacteriophage as disclosed above.
One aspect of the present invention relates to a composition comprising at least one bacteriophage selected from BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, or a variant thereof.
The present invention also relates to a composition comprising at least two different bacteriophages selected from BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, or variants thereof.
In a particular embodiment, the composition of the invention comprises BP1384 in combination with at least one other bacteriophage selected from BP1429, BP1430, BP1433, BP1450 or BP 1644.
In another particular embodiment, the composition of the invention comprises BP1384 in combination with at least one other bacteriophage selected from BP1450 and BP 1647.
In another particular embodiment, the composition of the invention comprises a combination of BP1430 and at least one other bacteriophage selected from BP1450, BP1644, BP1649 and BP 1661.
In another specific embodiment, the composition comprises BP1433 in combination with at least one other bacteriophage selected from BP1450, BP1647, BP1648, BP1650 and BP 1658.
In another preferred embodiment, the composition comprises BP1384 in combination with at least one other bacteriophage selected from BP1429, BP1647, BP1649 and BP 1662.
The present invention also relates to a composition comprising a combination of all phages BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, or variants thereof.
Specific examples of the composition of the present invention include:
the genome comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 4 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 5 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 9 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 4 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 10 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto, the genome comprising the nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 4 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto, the genome comprising the nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 5 or a sequence having at least 90% identity thereto; or
The genome comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto, the genome comprising the nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% identity thereto, and a bacteriophage whose genome comprises the nucleotide sequence of SEQ ID NO: 9 or a sequence having at least 90% identity thereto.
A particular embodiment of the present invention relates to a composition comprising:
the genome comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 2 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 4 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 5 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 6 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 7 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 8 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 9 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 10 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 11 or a sequence having at least 90% identity thereto;
the genome comprises SEQ ID NO: 12 or a sequence having at least 90% identity thereto; and
the genome comprises SEQ ID NO: 13 or a sequence having at least 90% identity thereto.
The compositions of the invention may also comprise other antibacterial agents, in particular other bacteriophages having different host specificities.
Preferred compositions of the invention have lytic activity against antibiotic-resistant strains of pseudomonas aeruginosa (p.
Other preferred compositions of the invention have lytic activity against more than 90% of all the bacterial strains of the LMG Collection obtained from the well-known BCCM/LMG bacterial Collection (BCCM/LMG Bacteria Collection). The depository can pass throughhttp://www.cabri.org/CABRI/srs-doc/bccm_lmg.info.htmlAnd accessing the website.
The antibacterial compositions of the present invention may be in a variety of different forms, such as liquid, semi-liquid, solid or freeze-dried formulations.
The compositions of the invention can comprise any effective amount of the selected bacteriophage. Preferably, they comprise 10e4To 10e12Between PFUs, preferably 10e5To 10e10PFU of each of the phages. The relative amounts of each type of bacteriophage in the compositions of the invention may be adjusted by the skilled artisan. Typically, when the antibacterial composition comprises several (n) different bacteriophages as defined above, the total relative amount of each bacteriophage in the composition,% a, is more preferably% a ═ 100/n (100/n)i) xV, wherein niRepresenting the number of different types of phages, V is a coefficient of variation between 0.2 and 5. Most preferably, V is between 0.3 and 3, even more preferably between 0.5 and 2, typically between 0.8 and 1.5. In a preferred exemplary embodiment, each type of bacteriophage is present in the composition of the present invention in approximately equal relative amounts.
The compositions of the invention preferably comprise a suitable diluent or carrier, for example a pharmaceutically or veterinarily acceptable excipient or carrier. In addition to the selected phage, the compositions of the present invention may include any excipient or carrier, such as thickeners, diluents, buffers, preservatives, surfactants, and the like. They include physiologically acceptable solutions or media that are not harmful to the organism or cause any significant specific or non-specific immune response or abolish the biological activity of the bacteriophage. For liquid formulations, saline, sterile water, ringer's solution, buffered saline, albumin infusion, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof may be used as pharmaceutically or veterinarily acceptable excipients or carriers. Other conventional additives such as thickeners, diluents, buffers, preservatives, surfactants, antioxidants and bacteriostats may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to the composition to prepare injectable preparations such as aqueous solutions, suspensions and emulsions, oral preparations such as pills, capsules, granules or tablets, or powder preparations. Formulations for topical administration may include bandages, dressings, patches, films, ointments, lotions, creams, gels, drops, suppositories, sprays, tampons, sanitary napkins, liquids and powders. Formulations for disinfection or for medical use may also include aerosols or sprays.
The compositions of the invention are useful in the medical field, including human or veterinary medicine, for example, for treating infections in mammals or for improving the condition of a subject. The compositions are useful for killing pseudomonas aeruginosa (p. aeruginosa) bacteria in an organism for the treatment of infections. The compositions may also be used to improve the condition of a mammal by improving the microbial flora in the mammal. In particular, the compositions of the invention can specifically remove strains of pseudomonas aeruginosa (p. aeruginosa) on mammalian skin or mucosa, thereby improving their microbial flora and restoring a suitable balance.
In a particular embodiment, the invention also relates to a method for treating an infection in a mammal, said method comprising administering to said mammal a composition or a bacteriophage or a nucleic acid or a polypeptide as defined above. In particular embodiments, the method comprises administering at least one, preferably at least two, even more preferably at least three, phages selected from BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, or variants thereof.
The invention also relates to the use of the described compositions, phages, nucleic acids or polypeptides for the manufacture of a medicament for the treatment of infections in mammals or for restoring the microbial flora in said mammals.
The compositions or agents of the invention may be administered by any convenient route, including intravenous, oral, transdermal, subcutaneous, mucosal, intramuscular, intrapulmonary, intranasal, parenteral, rectal, vaginal, and topical. In a preferred embodiment, the bacteriophage or composition is administered by a topical route, for example by application to the skin of a subject. The composition may be administered directly or indirectly, for example via a support. In this regard, the composition may be applied or sprayed, for example, to the affected area. The compositions of the invention may also be administered by oral or parenteral routes. The dosage suitable for administration, spraying or administration of the composition of the present invention may be adjusted by those skilled in the art according to various factors including formulation, mode of administration, age, body weight, sex, condition, diet, administration route and reaction sensitivity of the mammal to be treated at the time of administration. A physician of ordinary skill in the art can readily determine and prescribe the effective amount of the composition required.
The dosage may also be adjusted by the skilled artisan in order to obtain lytic activity against an antibiotic-resistant pseudomonas aeruginosa (p. Depending on the route of administration, an effective dose to obtain lytic activity in vivo will generally comprise at least 10e2PFU/ml, preferably about 10e2To 10e12Concentration of PFU/ml. Administration may be performed only once, or may be repeated if necessary.
The compositions of the present invention may be administered to treat pseudomonas aeruginosa (p. aeruginosa) infections, typically respiratory, urinary, burn, wound, ear, skin or soft tissue infections or gastrointestinal or post-surgical infections.
As shown in the experimental section, the bacteriophage and compositions of the present invention are capable of selectively killing pseudomonas aeruginosa (p. The composition can destroy a mixture of different pseudomonas aeruginosa bacteria even in vivo, even at low doses. In addition, the compositions of the present invention are effective in killing bacteria embedded in biofilms, which is particularly important for pathogenic bacteria. In addition, the compositions and phages of the invention are strictly unable to affect mammalian cells and are therefore specific in vivo and without side effects.
The invention also relates to the use of a composition, phage, nucleic acid or polypeptide of the invention for disinfecting a material. Due to their potent antibacterial effect and their ability to even compromise the integrity of bacterial biofilms, the compositions of the present invention can be used as disinfectants to eliminate bacteria on materials or at least cause a reduction in the number of bacteria. Such methods can be used to treat a variety of different biological or non-biological surfaces, including solid materials or devices such as contact lenses, surfaces of devices to be implanted into the body, tubing, catheters, laboratory vessels, fabrics, and the like, in both medical and non-medical contexts.
Diagnostic/prognostic assays of the invention:
the present invention also relates to a method for predicting or determining the efficacy of a phage therapy in a subject, wherein said method comprises the step of determining the lytic activity of one or more phages selected from the group consisting of BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662 on a pseudomonas aeruginosa (p. aeruginosa) strain from a sample of said subject, such lytic activity being indicative for an effective treatment. Preferably, the method further optionally comprises the step of treating said subject with one or more bacteriophages having lytic activity against a strain of pseudomonas aeruginosa (p.
In another aspect, the invention provides a method for selecting a subject or determining whether a subject is susceptible to benefit from phage therapy, wherein the method comprises the step of determining the lytic activity of one or more phages selected from BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662 on a pseudomonas aeruginosa (p.aeruginosa) strain from a sample of the subject, the lytic activity of one or more phages of the invention on at least one pseudomonas aeruginosa (p.aeruginosa) strain being indicative of a responder subject.
Another object of the invention relates to a method for predicting a subject's response to a phage therapy, wherein said method comprises the step of determining the lytic activity of one or more phages selected from BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662 against a pseudomonas aeruginosa (p.aeruginosa) strain from a sample of said subject, the lytic activity of one or more phages according to the invention against at least one pseudomonas aeruginosa (p.aeruginosa) strain indicating a good response to said therapy.
Other aspects and advantages of the present invention will be disclosed in the experimental section below, which is merely illustrative.
Examples
Materials and methods
Phage isolation and preparation
MDR pseudomonas aeruginosa (p. aeruginosa) bacteria were used to isolate and enrich each virulent phage from environmental water. Environmental samples were mixed with an overnight culture of bacteria in Luria Bertani (LB) and incubated at 37 ℃ with shaking for 24h to enrich for specific phages. At the end of the incubation, a few drops of chloroform were added to the culture. The culture was centrifuged at 11,000g for 5 minutes to remove bacterial cells and debris. The supernatant was passed through a 0.2 μm filter to remove residual bacterial cells. The enriched phage solution was plated on LB agar medium embedded with pseudomonas aeruginosa (p. Plaques formed on the plates after 24h incubation at 37 ℃. Individual plaques were picked for subsequent phage purification and amplification. The phage were then stored at 4 ℃ as a suspension in LB broth or physiological saline.
The titer of phage in suspension was estimated by plaque counting (Postic, 1961). A10-fold dilution of the suspension was delivered to the dry lawn of the propagated strain. Plates were read after overnight incubation. Plaque counting also allows visualization of plaque morphology.
Host range determination
The host range of the phage was determined in a panel of 20 pseudomonas aeruginosa (p. aeruginosa) from the LMG pool. Will 109The individual bacterial cells were mixed with the melted agar and the mixture was poured onto solid agar to make a double-layered agar plate. After curing, the isolated phage stock solution was spotted on each plate with a different bacterial strain. After allowing the spots to be absorbed for 20min, the plates were inverted and incubated at 27 ℃ for 24h, and then the extent of lysis was recorded (Postic, 1961; Yang, 2010).
Electron microscopy
An electron micrograph of each phage was taken using a transmission electron microscope.
Sequencing, analysis and annotation of phage genomes
To isolate phage DNA, the phage is propagated as described above. Phage DNA was purified by treatment with phenol: chloroform: isoamyl alcohol (25:24:1, V/V) extraction, ethanol precipitation and re-dissolution in water. Whole genome sequencing was performed and the BLAST algorithm was used to determine similarity to genes described in the National Center for Biotechnology Information [ NCBI ] database. The genome is scanned for potential Open Reading Frames (ORFs).
Example 1: phage-host characteristics and kinetics
One-step growth experiments were performed as described previously to determine first the productive lysis time, the adsorption rate and then the phage release amount. To determine the adsorption rate, samples were taken at different time intervals to analyze the solution for free phage particles. For determination of productivity time and amount of released phage, pseudomonas aeruginosa (p. aeruginosa) bacteria were mixed with phage solution and allowed to adsorb for 15 min. The mixture was immediately centrifuged at 5000rpm for 10min to remove free phage particles. The pellet was resuspended in 5 parts of fresh LB medium and the culture was incubated further at 37 ℃. Samples were taken at 3min intervals and phage titers were determined. These results allowed the calculation of the number of phages produced per bacterium (release), productive time and Productive Lytic Effect (PLE), as shown in table 5 below.
TABLE 4
These results show that all phages have strong virus production capacity and adsorption rate. Most phages had PLE below 7, confirming an outstanding characteristic. In this regard, bacteriophages 1429 and 1647 are particularly effective. Furthermore, the different PLE and adsorption times allow for the production of mixtures with selected variability.
Example 2: preparation of the Mixed composition
The following mixed compositions, each of which contains 10, were constructed-9To 10-11Each phage between pfu:
TABLE 5
| Mixture of | Bacteriophage |
| I | P1384+P1433 |
| II | P1384+P1450 |
| III | P1430+P1649 |
| IV | P1430+P1433 |
| V | P1430+P1650 |
| VI | P1384+P1430+P1433 |
| VII | P1384+P1430+P1450 |
| VIII | P1384+P1430+P1649 |
The following two other mixed compositions containing all the various bacteriophages were constructed, covering the most important diversity of the pseudomonas aeruginosa (p. aeruginosa) species:
mixed composition A：
Mixed composition B：
Example 3: sensitivity of bacteria to the phage mixture of the invention
Using the phage mixture of the invention, at 2.109The concentration of individual phages per ml was tested for various bacterial strains. Different bacterial concentrations were applied at a concentration of 2.109Individual phages per ml of phage mixture and incubation at 37 ℃ for 24 h.
The mixtures were tested on 22 different pseudomonas aeruginosa (p. aeruginosa) bacteria listed in tables 2 and 3. The% of bacterial species sensitive to the mixture are listed in table 6 below:
TABLE 6
| Mixture of | Killed P.aeruginosa species% |
| I | 73％ |
| II | 82％ |
| III | 91％ |
| IV | 86％ |
| V | 77％ |
| VI | 86％ |
| VII | 95％ |
| VIII | 95 |
| A | |
| 100 | |
| B | |
| 100％ |
The number of bacteria was calculated and used for the calculation of the resistance rate (number of bacteria after incubation/number of bacteria plated). The resistance rates using a mixture containing 13 different types of phages are shown in table 7 below:
TABLE 7
| Bacteria | Resistance Rate (bacteria/ml) |
| LMG 24891 | 1,00E-05 |
| LMG 24945 | 5,80E-06 |
| LMG 24970 | 1,00E-05 |
| LMG 25082 | 4,60E-06 |
| LMG 25131 | 9,00E-06 |
| LMG 25194 | 9,00E-06 |
All tested bacteria were sensitive to the composition of the present invention.
Example 4: specificity of the mixture
The specificity of the mixture was verified by testing on 10 bacterial species including Escherichia coli (Escherichia coli), Acinetobacter baumannii (Acinebacter baumannii), Enterobacter aerogenes (Enterobacter aerogenes), Enterobacter albugineus (Enterobacter albugineae), Enterobacter cloacae (Enterobacter cloacae), Klebsiella pneumoniae (Klebsiella pneumoniae), Proteus mirabilis (Portus mirabilis), Staphylococcus aureus (Staphylococcus aureus), Stenotrophomonas maltophilia (Stenotrophora), Serratia marcescens (Serratia marcescens).
Table 8 summarizes the lysis activity observed for each phage used alone or in combination as a mixture of 13 phages.
The above table clearly shows the absence of lytic activity against bacteria other than the pseudomonas aeruginosa (p. Thus, the bacteriophages and mixtures of the invention are highly specific for strains of pseudomonas aeruginosa (p.
Example 5: efficacy of phages on pseudomonas aeruginosa (p. aeruginosa) strains in vitro
Several strains of the LMG pool were selected to represent the genetic diversity and various forms of antibiotic resistance of pseudomonas aeruginosa (p. As set forth in the description of table 9,the strains are sensitive or resistant to one or several antibiotics. They were grown individually or in combination of 2 to 8 strains. Phage mixture 1 to 10e-6I.e., at a dilution rate of 1 to 1 million (bacteria/phage).
Table 9: information on bacterial strains
| LMG n° | State of the country | Year of year | Source | Serotype | ATB resistance type |
| LMG 24891 | France | 1882-1918 | Surgical bandage | 11 | 1 |
| LMG 24893 | Greece | 1994 | Sputum | 11 | 2 |
| LMG 24909 | Columbia | 2003 | Peritoneal fluid | 12 | 0 |
| LMG 24988 | Turkish for treatment of chronic gastritis | 1997 | Burn injury | 8 | 3 |
| LMG 24992 | Great Britain | 2003 | CF patients | NT | 4 |
| LMG 25041 | Philippines | 1993 | Wound healing device | NT | 2 |
| LMG 25049 | France | 1882-1918 | |
6 | 1 |
| LMG 25140 | Panama | 2006 | Wound healing device | 11 | 5 |
The results are shown in figure 1 and table 10 below.
Table 10: efficacy of the obtained phage mixture against pseudomonas aeruginosa (p. aeruginosa) mixture in vitro: at 2.10e7Density of cfu/ml and various dilutions:
the compositions of the present invention are capable of killing a mixture of 8 different pseudomonas aeruginosa (p. aeruginosa) bacterial strains together. At a dilution of 1/1000, the mixture was still effective against 8 strains.
Example 6: efficacy of bacteriophage on pseudomonas aeruginosa (p. aeruginosa) strains in vivo
The isolated Is580 strain collected in 1997 on burn patients was used in the following experiments.
The strain Is580 Is resistant to ampicillin, AMC, PIP, CEF, CXM acetoxyethyl ester, FOX, CPD, CTX, CAZ, GEN, TOB, OFX, NIT, SXT.
SKH1 mice (or hairless mice) were used as a mouse model for pseudomonas aeruginosa (p.
The method comprises the following steps: (see Table 11 below)
Immunosuppression of mice by a total of 3 injections of 1.5mg cyclophosphamide (Cy) IP every 2 days from day-3 before infection.
Burns on mouse skin with 2 μ l of liquid mustard gas at 30 mg/kg.
Two days after the burn, infection was performed by subcutaneous injection of a bacterial suspension at the burn site.
Table 11:
a mixed composition was prepared as in example 1 and applied on day 0 with 10e7Phage/ml phage mix soaked press cloth.
Various concentrations of pseudomonas aeruginosa (p. aeruginosa) strains were tested using 100 μ l phage cocktail. As shown on figure 2, all pseudomonas aeruginosa (p. aeruginosa) strains were killed 6h after treatment.
After administration of the Is580 pseudomonas aeruginosa strain to SKH1 mice by subcutaneous injection, only 35% of the mice survived in the absence of further treatment. In mice treated by injection of phage cocktail as shown in table 10 above, dramatic survival was observed (see figure 3): for subcutaneously treated SKH1 mice, 100% survived 16 days post infection. By way of comparison, for SKH1 mice treated with antibiotics, a survival rate of 50% was observed 2 days after infection.
Thus, the compositions of the invention can treat infections in vivo and can induce 100% survival in infected mice.
Reference to the literature
Clark WA,1962, Appl Microbiol. Comparison of several methods for preserving phages (Comparison of sectional methods for preserving bacteriophages), 1962 Sep; 10:466-71.
Drulis-Kawa Z, Majkowska-Skrobek G, macejewska B, Delattre AS, Lavigne R,2012, from bacteriophages — advantages and limitations of the use of bacteriophages and bacteriophage-encoded proteins (Learning from bacteriophages-additives and limitations of phase and phase-encoded protein applications); 13(8):699-722.
Fordos J.1859.Receuil des travaux de la Societéd'Emulation pour les Sciences Pharmaceutiques,vol 3Societéd'Emulation pour les Sciences Pharmaceutiques,Paris,France
Freeman L.1916. Pseudomonas aeruginosa caused Chronic systemic infection with the Bacillus pyoyana, Ann.Surg.64: 195-202.
Pseudomonas aeruginosa septicemia (Pseudomonas aeruginosa septicemia in Burns) in Burns, Burns 25: 611-.
Jones AM et al, 2010, clinical outcome of cystic fibrosis patients infected with p.aeruginosa: an 8-year prospective study (Clinical outer for Clinical fibers infected with a transmissible pathogenic agent: an 8-year productive study), check 137: 1405-1409.
Kang CI et al, 2005, bloodstream infection by antibiotic resistant gram negative bacilli: risk factors for mortality and the effect of inappropriate initial antimicrobial therapy on outcome (blood factors used by antimicrobial-resistant gram-negative bacteria: risk factors for movement and impact of inhibition of antimicrobial-resistant biological therapy on the output), and anti. agent chemistry. 49: 760-.
Micek ST et al, 2005, P.aeruginosa bloodstream infection: the importance of an appropriate initial antimicrobial therapy (Pseudomonas aeruginosa antibody infection: import of preprpriate antibiotic treatment), antibiotic. Agents Chemother.49: 1306-1311.
Strateva T. and Yordanov D.2009, Pseudomonas aeruginosa-bacterial resistance phenomenon (Pseudomonas aeruginosa-a phenomenon of bacterial resistance), Journal of Medical Microbiology 58, 1133-.
The Ecology of Weinbauer MG. prokaryotic viruses (Ecology of prokarstic viruses), FEMS Microbiol Rev 2004; 28:127-81.
Williams EP, Cameron K.1894 Pseudomonas aeruginosa Infection-a cause of infant death (Infection by the Bacillus pyocyaneus a house of infant mortality), Public Health Page.Rep.20: 355-.
Claims (21)
1. An antibacterial composition comprising at least two different bacteriophages having lytic activity against a strain of pseudomonas aeruginosa (p. aeruginosa) selected from the group consisting of bacteriophages having genomes consisting of SEQ ID NO: 1 to 13, or a pharmaceutically acceptable salt thereof.
2. The composition of claim 1, comprising at least three different bacteriophages selected from the group consisting of phages having genomes represented by SEQ ID NO: 1 to 13, or a pharmaceutically acceptable salt thereof.
3. The composition of claim 1, comprising at least four different bacteriophages selected from the group consisting of phages having genomes consisting of SEQ ID NO: 1 to 13, or a pharmaceutically acceptable salt thereof.
4. The composition of claim 1, comprising any one of the following phage combinations:
the genome consists of SEQ ID NO: 1, and a genome consisting of the nucleotide sequence of SEQ ID NO: 4, or a bacteriophage consisting of the nucleotide sequence of seq id no; or
The genome consists of SEQ ID NO: 1, and a genome consisting of the nucleotide sequence of SEQ ID NO: 5, or a bacteriophage consisting of the nucleotide sequence of seq id no; or
The genome consists of SEQ ID NO: 3, and the genome consists of the nucleotide sequence of SEQ ID NO: 9, a bacteriophage consisting of the nucleotide sequence of seq id no; or
The genome consists of SEQ ID NO: 3, and the genome consists of the nucleotide sequence of SEQ ID NO: 4, or a bacteriophage consisting of the nucleotide sequence of seq id no; or
The genome consists of SEQ ID NO: 3, and the genome consists of the nucleotide sequence of SEQ ID NO: 10, or a bacteriophage consisting of the nucleotide sequence of seq id no; or
The genome consists of SEQ ID NO: 1, and a genome consisting of the nucleotide sequence of SEQ ID NO: 3, and the genome consists of the nucleotide sequence of SEQ ID NO: 4, or a bacteriophage consisting of the nucleotide sequence of seq id no; or
The genome consists of SEQ ID NO: 1, and a genome consisting of the nucleotide sequence of SEQ ID NO: 3, and the genome consists of the nucleotide sequence of SEQ ID NO: 5, or a bacteriophage consisting of the nucleotide sequence of seq id no; or
The genome consists of SEQ ID NO: 1, and a genome consisting of the nucleotide sequence of SEQ ID NO: 3, and the genome consists of the nucleotide sequence of SEQ ID NO: 9, or a bacteriophage consisting of the nucleotide sequence of seq id No. 9.
5. The composition of claim 1, comprising a combination of all bacteriophages BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and BP1662, which are represented by SEQ ID NOs: 1 to 13.
6. The composition of any one of claims 1 to 5, which has lytic activity against an antibiotic-resistant Pseudomonas aeruginosa strain.
7. The composition of any one of claims 1 to 5, which has lytic activity against more than 90% of all bacterial strains of the LMG deposit.
8. The composition of any one of claims 1 to 5, further comprising a pharmaceutically acceptable excipient or carrier.
9. The composition of any one of claims 1 to 5, which is a liquid, semi-liquid, solid or lyophilized formulation.
10. The composition of any one of claims 1 to 5 comprising 10e4To 10e12Each phage between PFUs.
11. Use of a composition according to any one of claims 1 to 10 in the manufacture of a medicament for treating an infection in a mammal.
12. Use of a composition of any one of claims 1 to 10 in the manufacture of a medicament for improving the condition of a mammal by improving the microbial flora in said mammal.
13. In vitro use of a composition according to any one of claims 1 to 10 for disinfecting a material.
14. A process for preparing a composition according to any one of claims 1 to 10, said process comprising separately producing said one or more bacteriophages and combining said bacteriophages with a suitable carrier or excipient.
15. Use of any one or more bacteriophage as defined in any one of claims 1 to 10 in the manufacture of a medicament or kit for predicting or determining the efficacy of a bacteriophage therapy against pseudomonas aeruginosa infection, said therapy comprising said one or more bacteriophage.
16. A bacteriophage having lytic activity against a pseudomonas aeruginosa (p. aeruginosa) strain, and the genome of said bacteriophage consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 13, or a pharmaceutically acceptable salt thereof.
17. An isolated nucleic acid consisting of a sequence selected from the group consisting of SEQ ID NO: 1 to 13, or a pharmaceutically acceptable salt thereof.
18. A composition comprising the bacteriophage of claim 16 or the nucleic acid of claim 17.
19. Use of a bacteriophage of claim 16 or a nucleic acid of claim 17 or a composition of claim 18 in the manufacture of a medicament for treating an infection in a mammal.
20. Use of a bacteriophage of claim 16 or a nucleic acid of claim 17 or a composition of claim 18 in the manufacture of a medicament for improving the condition of a mammal by modifying the microbial flora in said mammal.
21. An in vitro use of a bacteriophage of claim 16 or a nucleic acid of claim 17 or a composition of claim 18 for disinfecting a material.
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| EP3584314A4 (en) * | 2017-02-17 | 2020-09-16 | Intron Biotechnology, Inc. | Novel pseudomonas aeruginosa bacteriophage pse-aep-3 and use thereof for inhibiting proliferation of pseudomonas aeruginosa |
| EP3372085A1 (en) | 2017-03-08 | 2018-09-12 | Pherecydes Pharma | Phage therapy |
| US10574580B2 (en) * | 2017-07-04 | 2020-02-25 | Vmware, Inc. | Network resource management for hyper-converged infrastructures |
| CA3088890A1 (en) * | 2018-01-02 | 2019-07-11 | Armata Pharmaceuticals, Inc. | Bacteriophage compositions for treating pseudomonas infections |
| CN108070572A (en) * | 2018-01-25 | 2018-05-25 | 青岛诺安百特生物技术有限公司 | A kind of width fragmentation pattern pyocinophages and its disinfection application |
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| CA3213424A1 (en) * | 2021-03-30 | 2022-10-06 | Lior Moshe ZELCBUCH | Pseudomonas bacteriophage and uses thereof |
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Effective date of registration: 20240417 Address after: lyon Patentee after: Airuitai Pharmaceutical Co. Country or region after: France Address before: Fa Guoluomanweier Patentee before: PHERECYDES PHARMA Country or region before: France |